In the study of biological and biochemical samples, fluorescent labels are often used as markers for species of interest. The fluorescent label is typically a small molecule with ring structures or a genetically encoded fluorescent protein, although in some cases the intrinsic fluorescence of a species already present in the sample may be used as the fluorescent label. These labels are detected by means of photons emitted when the labels relax from an excited state to a ground state. A process of repeated excitation-emission tends to result in “photobleaching” whereby the labels lose their ability to fluoresce and may produce a species damaging to the sample under study.
The process of photobleaching is thought to involve transition of photons into a bleached state via a dark (or triplet) state. It appears that this dark state is long-lived and is susceptible to bleaching, as illustrated in FIG. 1. Different fluorescent labels exhibit different emission and bleach profiles. Increasing the bleaching rate reduces the ability to image a sample over long periods of time, particularly when using a relatively noisy detection system.
Scientists studying biological systems are interested in observing samples over periods of time and with enough spatial and temporal resolution to distinguish events of interest. These requirements place considerable demands on the performance of the fluorescent labels and in particular their resistance to photobleaching under an often intense excitation beam. The bleaching rate is therefore often a significant factor in the selection of a suitable fluorescent label.
Fluorescence microscopy may image a sample using a wide-field format. Alternatively, a confocal imaging system may be used, in which an arrangement of optics and matching pinholes restrict the light so as to focus on a particular plane. A common implementation is the “Laser Scanning Confocal Microscope” (LSCM). In this design, a single intense beam of light is scanned across a sample and the resulting fluorescence detected using a photomultiplier tube. These systems expose the sample, and thus the fluorescent labels, to very intense light for short periods of time in order to scan the entire field of view. For example, an exposure time of 4 microseconds may be used in order to obtain a 500×500 pixel image in one second. This often results in rapid photobleaching of the fluorescent labels.
An alternative approach is to scan the sample with a slit rather than a pinhole. This allows more light to be used to illuminate the sample at the cost of reduced confocal sectioning ability.
A further approach is to apply a scanning protocol to scan a number of pinholes in parallel to create an image on an array detector such as a CCD. This may be achieved using a spinning disk, whereby one or more perforated disks are rotated between the excitation light source and the sample, such that each point on the same receives a brief burst of light as the disk(s) rotate, with many distinct points excited at any one time. Alternatively, the pinhole may be scanned in the x and y directions to build up an image.
In a related approach, a set of pinholes is created and scanned using a controllable array of optical elements to synthesise an array of pinholes. This approach is sometimes called “Programmable Array Microscopy” (PAM). Each element is under computer control and may be switched to emulate the pinhole disc without any large moving parts [Hagen 2007].
For example when the optical element is a controllable mirror switchable to an angle of 0 degrees or 10 degrees (such as in the Texas instruments Digital Micro-mirror Device), the optical path is arranged so that the light from the excitation source passes to the array and may be reflected onto the sample at each point when the mirror is switched on, or away from the sample when the mirror is switched off. Similarly, the optical element may be a controllable light cell switchable to be transparent or dark (such as in a spatial light modulator), and the optical path is arranged so that the light from the excitation source passes through the array before reaching the sample at each point when the light cell is switched on, or not reaching the sample when the light cell is switched off.
Another technique for scanning a sample with excitation light involves provision of an array of individual light sources such as light emitting diodes (LEDs). Each element is under computer control and may be switched to emulate the pinhole disc without any moving parts [Poher 2007].
The presence of molecular oxygen in a sample has been found to increase the rate at which bleaching takes place. One approach designed to reduce this bleaching is to purge the oxygen with another gas such as nitrogen. This has been found to be applicable to simply biochemical systems such as samples spin-coated on slides.
Alternatively, the use of a chemical oxygen scavenger system has been suggested, for example an enzymatic system of glucose oxidase and catalase. This may be supplemented with a triplet-state quencher [Rasnik 2006]. This is suitable for cell-free systems, but in many samples, the oxygen plays an important role and so it may not be possible to remove it without interfering with the processes of interest.
Another proposal for reducing photobleaching is to reduce the light dose received by the sample by modulating the excitation according to the detected signal. This is on the basis that the brightest regions need less excitation, and dark areas do not need to be exposed. This has been referred to as “Controlled Light Exposure Microscopy” (CLEM) [Hoebe 2007].
In many fluorescence microscopes, an illumination option is available in which a dye of interest is bleached intentionally. This provides a means of studying mobility rate, bound fractions, binding rates and so on using a set of related techniques often referred to as “Fluorescence Recovery After Photobleaching” (FRAP). The aim is to bleach the dye as effectively as possible. This may be achieved by imaging the sample at an increased light intensity which eventually bleaches the dye.
In another technique, the ability to accurately localise molecules labelled with a fluorescent dye may be estimated using numerically fitting techniques to process signals representing detected radiation. By selective bleaching a certain proportion of the labels, the chance of detecting isolated fluorescent dye molecules is enhanced. Accumulating sets of images with different subsets of bleached and unbleached molecules allows a form of ultra-high resolution to be completed with considerably improved resolving power relative to that available using bulk imaging of all dye molecules. Two such schemes have been published: “Photo-activated Localisation Microscopy” (PALM) [Betzig 2006] and “Sub-Defraction-Limit Imaging By Stochastic Optical Reconstruction Microscopy” (STORM) [Rust 2006]. These techniques are assisted by the use of photo-activation or transient photobleaching which allow different subsets of molecules to be detected and localised in each group of images in multiple cycles of imaging and selective bleaching.